During the past year, this laboratory continued studies on the mechanism of HIV entry, and on the development of novel protective and treatment strategies based on the molecules involved in entry. 1) Molecular mechanisms of gp120 interactions with coreceptors (CCR5 and CXCR4). a) Synthetic peptides derived from the predicted extracellular loops (ECLs) of CCR5 shown to neutralize HIV fusion and infection. The activities were shown to reflect binding of these peptides to gp120. These studies provide support for designing a soluble form of CCR5 capable of interacting specifically with the relevant regions of gp120 (collaboration with K. Ridge); they also suggest the possibility of structural studies to elucidate structure/function relationships in gp120 binding to CCR5 (collaborations under discussion). b) First demonstration that for gp120 molecules from genetically diverse strains, there is a strong correlation between coreceptor usage and sensitivity to neutralization by a particular anti-V3 loop MAb (D19). In the native surface trimer of CCR5-specific strains, the D19 epitope is masked prior to CD4 binding, rendering them resistant to D19 neutralization; by contrast, the epitope is constitutively exposed on CXCR4-using strains, rendering them neutralization sensitive. These results suggest the possibility that antibodies may exert selective pressures against CXCR4 usage, thereby contributing to absence of CXCR4-using strains in healthy infected people. c) Conceived and began testing a specific model for gp120 interactions with CCR5, based on known dynamic features of these molecules. 2) Novel anti-HIV agents based on HIV Env/receptor interactions. a) Topical microbicide to protect against sexual transmission of HIV. We previously designed a recombinant bifunctional protein, designated sCD4-17b, that neutralizes HIV-1 infection with extremely high potency. In a collaboration with OSEL, Inc., we are engineering vaginal strains of Lactobacillus to produce sCD4-17b. Upon successful colonization of the vaginal mucosa, the engineered bacteria would hopefully function as a "live microbicide" to neutralize virus introduced during intercourse. During the past year, we made modifications in the sCD4-17b construct that greatly reduced the previously observed problem of proteolysis (mainly within the linker regions). We are also testing various strategies to enhance production and secretion of the protein (testing of different promoters, stable integration into the bacterial genome, etc.). As related approachs, we have initiated collaborations with Dr. Dean Hamer (NCI) to engineer Lactobacillus for production of a different class of HIV-neutralizing agents, namely T20-based peptides which are already in use for salvage therapy in infected people who are failing HAART, as well as to engineer E. coli to secrete sCD4-17b for protection against rectal transmission of HIV. b) Anti-HIV immunotoxin to deplete reservoirs of HIV-infected cells. Through an NIH license with IVAX, production of clinical grade 3B3-PE38 is underway. Phase I clinical trials are being discussed with several potential collaborators.